VEGFD signaling balances stability and activity-dependent structural plasticity of dendrites

Mature neurons have stable dendritic architecture, which is essential for the nervous system to operate correctly. The ability to undergo structural plasticity, required to support adaptive processes like memory formation, is still present in mature neurons. It is unclear what molecular and cellular processes control this delicate balance between dendritic structural plasticity and stabilization. Failures in the preservation of optimal dendrite structure due to atrophy or maladaptive plasticity result in abnormal connectivity and are associated with various neurological diseases. Vascular endothelial growth factor D (VEGFD) is critical for the maintenance of mature dendritic trees. Here, we describe how VEGFD affects the neuronal cytoskeleton and demonstrate that VEGFD exerts its effects on dendrite stabilization by influencing the actin cortex and reducing microtubule dynamics. Further, we found that during synaptic activity-induced structural plasticity VEGFD is downregulated. Our findings revealed that VEGFD, acting on its cognate receptor VEGFR3, opposes structural changes by negatively regulating dendrite growth in cultured hippocampal neurons and in vivo in the adult mouse hippocampus with consequences on memory formation. A phosphoproteomic screening identified several regulatory proteins of the cytoskeleton modulated by VEGFD. Among the actin cortex-associated proteins, we found that VEGFD induces dephosphorylation of ezrin at tyrosine 478 via activation of the striatal-enriched protein tyrosine phosphatase (STEP). Activity-triggered structural plasticity of dendrites was impaired by expression of a phospho-deficient mutant ezrin in vitro and in vivo. Thus, VEGFD governs the equilibrium between stabilization and plasticity of dendrites by acting as a molecular brake of structural remodeling. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05357-2.

ESARE>LacZ-HA with or without Bic treatment for 24 h.c QRT-PCR analysis of VEGFD mRNA expression in cultured hippocampal neurons infected with rAAV-ESARE>VEGFD-HA or rAAV-ESARE>LacZ-HA with or without Bic treatment for 24 h.One-way ANOVA followed by Bonferroni's post hoc test.N = 5 independent culture preparations.d Proximity ligation assay (PLA) with antibodies against VEGFR3 and phospho-tyrosine of cultured hippocampal neurons with or without rVEGFD treatment for 2 h.Left side shows representative images of PLA signal in neurons treated as indicated.Nuclei were labelled with Hoechst.Scale bar = 20 µm.Lower panels show higher magnification of dendrites labeled with MAP2.Scale bar = 5 µm.Right side shows PLA cluster number normalized on the number of Hoechst-labelled cells and on control.One sample t-test.N = 5 independent culture preparations.e-g Morphometric analyses of cultured hippocampal neurons transfected with hrGFP, with or without rVEGFD, rVEGFD-Elab (rVEGFD-Alt) or Bic treatment, or in combination for 24 h.e Representative images of neurons treated as indicated.hrGFP was used to visualize neurons.Scale bar = 20 µm.f, g Total dendritic length (f) and total number of intersections (g) of neurons treated as indicated.One-and Two-way ANOVA followed by Bonferroni's post hoc test.N = 11-12 neurons from 3 independent culture preparations.
h Morphometric analyses (total dendritic length) of cultured hippocampal neurons transfected with hrGFP, with or without rVEGFD, DC101 or Bic treatment, or in combination for 24 h.One-way ANOVA followed by Bonferroni's post hoc test N = 11-12 neurons from 3 independent culture preparations.
i Morphometric analyses (total dendritic length) of cultured hippocampal neurons transfected with hrGFP, with or without rVEGFD, rVEGFA or Bic treatment, or in combination for 24 h.One-way ANOVA followed by Bonferroni's post hoc test N = 12 neurons from 3 independent culture preparations.j Morphometric analyses (total dendritic length) of cultured hippocampal neurons transfected with hrGFP, with or without rVEGFD, SAR131675 or Bic treatment, or in combination for 24 h.One-way ANOVA followed by Bonferroni's post hoc test N = 11-12 neurons from 3 independent culture preparations.k-n Morphometric analyses of cultured hippocampal neurons transfected with hrGFP and with or without rVEGFC or Bic treatment, or both for 24 h.k Representative images of neurons transfected and treated as indicated.hrGFP was used to visualize neurons.Scale bar = 20 µm.l-n Total dendritic length (l), Sholl analysis (minimal significant p value is displayed) (m) and total number of intersections (n) of neurons treated as indicated.One-and Two-way ANOVA followed by Bonferroni's post hoc test (l, n), or Dunnett's post hoc test (m).N = 14-17 neurons from 3 independent culture preparations.o, p Time-lapse imaging of neurite length for 24 h of hrGFP-transfected cultured hippocampal neurons with or without rVEGFC or Bic treatment, or both.o Schema of the experimental setup.p Relative total neurite length over time normalized on the first timepoint and untreated control.Two-way ANOVA followed by Dunnett's post hoc test.N = 3 independent cultures.a, b Immunoblot analysis in cultured hippocampal neurons treated with DMSO (vector) or 1 μM or 10 μM PP2 for 1 h or 24 h. a Representative immunoblots of ezrin, phospho Y478 ezrin, phospho Akt and tubulin.b Phospho Akt, ezrin, phospho Y478 ezrin and its phospho/total protein ratio normalized on tubulin and control.One sample t-test.N = 3-5 independent culture preparations.c, d Proximity ligation assay (PLA) with antibodies against ezrin and phospho Y478 ezrin in cultured hippocampal neurons treated with DMSO (vector) or 10 μM PP2 for 1 h.c Representative images of PLA signal in neurons treated as indicated.Nuclei were visualized with Hoechst.Scale bar = 20 μm.d PLA cluster number normalized on the number of Hoechst-labelled cells and on control.One sample t-test.N = 5 independent culture preparations.
e Relative phosphorylation level of phospho Ser75, Tyr216, Tyr 418 and Tyr529 Src derived from the cytoskeleton-based phosphoantibody array from cultured hippocampal neurons with or without treatment of rVEGFD for 15 min or 2h (see also main figure 6).One-way ANOVA followed by Dunnett's post hoc test.N = 3 independent culture preparations.f, g Immunoblot analyses of phospho Src protein expression in cultured hippocampal neurons with or without treatment of rVEGFD for 15 min.f Representative immunoblots of pSrc and tubulin.pSrc (g) levels normalized to tubulin expression and control.One sample t-test.N = 4 independent culture preparations.h, i Immunoblot analyses of phospho Src protein expression in cultured hippocampal neurons with or without treatment of rVEGFD for 2 or 24 h.h Representative immunoblots of pSrc and tubulin.i pSrc levels normalized to tubulin expression and control.One-way ANOVA followed by Dunnett's post hoc test.N = 4 independent culture preparations.
After 5 min of baseline recording, neurons were treated with 50 µM Bic or 100 ng/ml rVEGFD and 50 µM Bic and imaged for another 10 min.Data were collected using proprietary software (VisiView, Visitron Systems), and analyzed using Fiji and IgorPro (WaveMetrics).Calcium signals in regions of interest drawn around individual nuclei were quantified as the baseline intensity-normalized change in fluorescence over time, ΔF/F = (F−Fbaseline)/Fbaseline.Calcium transients were quantified in terms of the mean peak amplitude and the mean inter-peak interval following the start of the treatment.

Calcineurin activity
Calcineurin activity was measured on lysates of cultured neurons using a specific Calcineurin Cellular Activity Assay Kit (Enzo Life Sciences) according to the manufacturer's protocol.In brief, after treatments, cultures were harvested in lysis buffer containing proteases inhibitors.Before proceeding with the assay, samples were checked for the presence of free phosphate which may affect the subsequent measurements.The method relies on the RII phosphopeptide substrate -known for its specificity for calcineurin-and measures the amount of released phosphate from the substrate via the Malachite green assay.Recombinant calcineurin was included as positive control.A calibration curve was created for each measurement, and absorbance values were used to calculate the amount of free phosphate from a standard curve line-fit in Prism software.

a
Graphs represent mean ± SEM.Dots represent single values.Asterisks (*) refer to statistical comparisons between conditions and hashtags (#) to comparisons to basal values per condition.****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ####p < 0.0001; ###p < 0.001; ##p < 0.01; #p < 0.05; ns p>0.05 Supplementary fig. 2 Nuclear calcium transients induced by neuronal activity are not affected by VEGFD.Representative images of jRGECO1alpha-NLS fluorescence in cultured hippocampal neurons prior to and after bicuculline (Bic) or Bic and rVEGFD administration.b Representative traces showing the time course of evoked Bic-induced nuclear calcium transients in neurons treated as indicated.Scale bar = 100 µm.c, d Mean inter-peak interval (c) and mean peak amplitude (d) during the first ten minutes following treatment.Two-tailed unpaired Student's t-test.N = 6-7 coverslips from 4 independent culture preparations.Graphs represent mean ± SEM.Dots represent single values Supplementary fig. 3 Src is not involved in VEGFD-mediated regulation of Y478 ezrin phosphorylation in neurons.

j, k
Immunoblot analyses of phospho NFATc1 protein expression in cultured hippocampal neurons with or without treatment of rVEGFD for 15 min.j Representative immunoblots of phospho NFATc1 and tubulin.k Phospho NFATc1 levels normalized to tubulin expression and control.One sample t-test.N = 3 independent culture preparations.l Free phosphate released by a phosphopeptide substrate and indicative of calcineurin activity in homogenates from cultured hippocampal neurons with or without treatment of rVEGFD for 15 min.Two-tailed unpaired Student's t-test.N = 3 independent culture preparations.Graphs represent mean ± SEM.Dots represent single values.***p < 0.001; *p < 0.05 Supplementary fig. 4 Overexpression of ezrin affects VEGFD and VEGFR3 expression.a-b QRT-PCR analysis of VEGFD (a) and VEGFR3/Flt4 (b) mRNA expression levels in cultured hippocampal neurons infected with rAAV-LacZ, rAAV-ezrin WT or rAAV-ezrin Y478F.Values are normalized to LacZ-infected cultures.One-way ANOVA followed by Dunnett's post-hoc test.N = 5 independent culture preparations.Graphs represent mean ± SEM.Dots represent single values.* p<0.05; **** p< <0.0001; ns p>0.05